Dried blood samples are increasingly being used as an alternative for classical venous blood and plasma. In the analysis of dried blood samples, a major ‘unknown’, having an impact on the uncertainty of the analytical result, is the hematocrit (Hct), i.e. the volume percentage of red blood cells in blood.
As the Hct cannot be directly measured on dried blood samples, people have tried to estimate this based on the levels of endogenous compounds that correlate with the Hct.
These attempts have for example been reviewed in De Kesel et al., 2013 (Bioanalysis 2013; 5(16):2023-41).
One possible method was to use potassium as a value for red blood cell content, such as reported by Capiau et al., 2013 (Anal Chem. 2013; 85(1):404-10). Although this method showed good results, one drawback is that part of the dried blood sample needs to be sacrificed for the potassium measurement, which is undesirable.
In view of implementation of this method in high-throughput surroundings such as clinical labs or pharmaceutical industry, it is furthermore important that a Hct estimation method is automatable and compatible with existing automated dried blood sample analysers. Furthermore, this method should preferably be non-destructive, as this way no part of the very limited sample volume is consumed and the entire dried blood sample remains available for further analyses.
Another logical candidate Hct marker is hemoglobin, as hemoglobin is also used for this purpose in whole blood samples. However, multiple research groups have tried this and failed. E.g. Miller et al., 2013 (J Anal Bioanal Techniques) report on a method to estimate hematocrit in a dried blood spot comprising measuring hemoglobin via non-contact diffuse reflectance spectroscopy at a wavelength of 980 nm. The reflection obtained at this wavelength is not representative for the hemoglobin in the dried blood sample, but reflects the background scattering of the substrate, which is not specific for hemoglobin. Miller et al further state that although they tried to use the hemoglobin-specific parts of the spectrum (using wavelengths of 540 and/or 570 nm) as a measure for Hct, they did not succeed. Yu and co-workers (Anal. Bioanal. Chem., 2015 Sep. 7) recently reported on another method of quantifying hemoglobin in dried blood spots using a protein spiking method coupled to detection with tandem mass spectrometry; One drawback of this method is that the sample is destroyed and hence cannot be used for further analyte analysis.
Hence, up till now, hemoglobin was considered not suitable for the prediction of the Hct of dried blood samples.
Accordingly, there is a need for improved methods for determining the hematocrit in a dried blood sample, which mitigate at least one of the problems stated above.